Adenoviruses belong to the family Adenoviridae, which is divided into two genera, namely Mastadenovirus and Aviadenovirus. Adenoviruses are nonenveloped, regular icosahedrons 65-80 nm in diameter (Horne et al., J. Mol. Biol., 1, 84-86 (1959)). The capsid is composed of 252 capsomeres of which 240 are hexons and 12 are pentons (Ginsberg et al., Virology, 28, 782-783 (1966)). The hexons and pentons are derived from three different viral polypeptides (Maizel et al., Virology, 36, 115-125 (1968); Weber et al., Virology, 76, 709-724 (1977)). The hexon comprises three identical polypeptides of 967 amino acids each, namely polypeptide II (Roberts et al., Science, 232, 1148-1151 (1986)). The penton contains a penton base, which is bound to the capsid, and a fiber, which is noncovalently bound to and projects from the penton base. The fiber protein comprises three identical polypeptides of 582 amino acids each, namely polypeptide IV. The Ad2 penton base protein is an 8.times.9 nm ring-shaped complex composed of five identical protein subunits of 571 amino acids each, namely polypeptide III (Boudin et al., Virology, 92, 125-138 (1979)). Proteins IX, VI, and IIIa are also present in the adenovirus coat and are thought to stabilize the viral capsid (Stewart et al., Cell, 67, 145-154 (1991); Stewart et al., EMBO J., 12(7), 2589-2599 (1993)) .
Once an adenovirus attaches to a cell, it undergoes receptor-mediated internalization into clathrin-coated endocytic vesicles of the cell (Svensson et al., J. Virol., 51, 687-694 (1984); Chardonnet et al., Virology, 40, 462-477 (1970)). Virions entering the cell undergo a stepwise disassembly in which many of the viral structural proteins are shed (Greber et al., Cell, 75, 477-486 (1993)). During the uncoating process, the viral particles cause disruption of the cell endosome by a pH-dependent mechanism (Fitzgerald et al., Cell, 32, 607-617 (1983)), which is still poorly understood. The viral particles are then transported to the nuclear pore complex of the cell (Dales et al., Virology, 56, 465-483 (1973)), where the viral genome enters the nucleus, thus initiating infection.
An adenovirus uses two separate cellular receptors, both of which must be present, to attach to and efficiently infect a cell (Wickham et al., Cell, 73, 309-319 (1993)). First, the fiber protein attaches the virus to a cell by binding to an, as yet, unidentified receptor. Then, the penton base binds to .alpha..sub.v integrins. Integrins are a family of heterodimeric cell surface receptors that mediate cellular adhesion to the extracellular matrix molecules fibronectin, vitronectin, laminin, and collagen, as well as other molecules (Hynes, Cell, 69, 11-25 (1992)). Integrins are known to play important roles in cell signaling processes, including calcium mobilization, protein phosphorylation, and cytoskeletal interactions (Hynes, supra). The specificity with which an integrin binds to a particular ligand, such as those associated with an adenovirus, is a function of the paired .alpha. and .beta. subunits of the integrin. For example, integrin .alpha..sub.6 .beta..sub.1 binds to laminin, integrin .alpha..sub.2 .beta..sub.1 binds to collagen and laminin, and integrin .alpha..sub.3 .beta..sub.1 binds to collagen, laminin, and fibronectin. Furthermore, different tissue types may have different complements of integrin subunits, thereby providing a mode of spatial control over integrin-ligand signal transfer or over ligand internalization. Accordingly, some integrins, such as those that include subunit .alpha..sub.v, are broadly expressed on numerous cell types, whereas other integrins have a much more narrow tissue distribution. For example, the integrins that include subunit .beta..sub.2 are expressed only on leukocytes, such as neutrophils and macrophages, integrins including subunit .alpha..sub.4 are expressed only on lymphocytes and fibroblasts, and the integrin defined by the subunits .alpha..sub.IIb 62 .sub.3 is expressed only on platelets and megakaryocytes.
The specificity of integrin subunit complement also extends to the infectability of cells by different serotypes of adenovirus, because the particular .alpha. and .beta. subunits dictate whether a virus can enter a cell. For example, the penton base of the adenovirus serotype Ad2 binds to integrins .alpha..sub.v .beta..sub.3 and .alpha..sub.v .beta..sub.5 (Wickham et al. (1993), supra). Given that both receptors utilized by an adenovirus are expressed on most human cells, nearly all cells in a human body are susceptible to adenoviral infection.
A majority of integrins have been found to recognize short linear stretches of amino acids in binding to a specific ligand. The tripeptide motif arg-gly-asp (RGD) [SEQ ID NO:1], which is found in scores of matrix ligands, including laminin, fibronectin, collagen, vitronectin, and fibrinogen, has been implicated in the binding of .alpha..sub.3 .beta..sub.1, .alpha..sub.5 .beta..sub.1, .alpha..sub.IIb .beta..sub.3, .alpha..sub.m .beta..sub.2, and most, if not all, of the five .alpha..sub.v -containing integrins. The conformation of the RGD sequence within a matrix ligand is thought to be a primary factor in integrin specificity (Pierschbacher et al., J. Biol. Chem., 262, 17294-17298 (1987)). Sequences that directly flank the RGD sequence have been shown to influence integrin specificity, presumably because of their effect on RGD conformation (Smith et al., Proc. Natl. Acad. Sci. USA, 90, 10003-10007 (1993)). However, sequences distant from the RGD may also function in integrin specificity as has been shown for the binding of .alpha..sub.5 .beta..sub.1 to fibronectin (Obara et al., Cell, 53, 649-657 (1988)). Other integrins, which do not utilize RGD, have been found to bind similar short linear stretches of amino acids within their specific ligands. For example, integrin .alpha..sub.IIb .beta..sub.3 binds via the amino acid sequence lys-gln-ala-gly-asp (KQAGD) [SEQ ID NO:2] in fibrinogen (Kloczewiak et al., Biochemistry, 23, 1767-1774 (1984)), while .alpha..sub.4 .beta..sub.1 binds via the core sequence glu-ile-leu-asp-val (EILDV) [SEQ ID NO:3] in fibronectin (Komoriya et al., J. Biol. Chem., 266, 15075-15079 (1991)). A structural motif (i.e., asn-pro-xaa-tyr (NPXY) [SEQ ID NO:4]) present in the .beta. subunits of .alpha..sub.v -containing integrins has been shown to be important for internalization (Suzuki et al., Proc. Natl. Acad. Sci. USA, 87, 5354 (1990)).
The penton base sequence is highly conserved among serotypes of adenovirus and contains five copies of the RGD tripeptide motif (Neumann et al., Gene, 69, 153-157 (1988)). The RGD tripeptide is believed to mediate binding to .alpha..sub.v integrins because exogenously added RGD peptides can block penton base binding and adenoviral infection (Wickham et al. (1993), supra), and adenoviruses that have point mutations in the RGD sequence of the penton base are restricted in their ability to infect cells (Bai et al., J. Virol., 67, 5198-5205 (1993)).
The penton base genes from Ad2, Ad5, Ad12, and Ad40 serotypes of adenovirus have been sequenced. Alignment of the sequences reveals a high degree of conservation over the entire sequence, except for the N-terminus, and, in Ad2, Ad5, and Ad12, a hypervariable region that includes the RGD sequence. Only Ad40, one of two enteric adenoviral serotypes, does not have an RGD sequence. Ad2 and Ad5 are identical in the hypervariable region and contain a large insert of amino acids flanking either side of the RGD sequence. Secondary structural analysis of the hypervariable regions of the three RGD-containing penton bases predicts that, in each case, the RGD is flanked by .alpha.-helices. Such structures are believed to form the spikes seen in cryo-electron micrographic (cryo-EM) images of Ad2 penton bases (Stewart et al. (1993), supra).
Recombinant adenoviral vectors have been used for the cell-targeted transfer of one or more recombinant genes to diseased cells or tissue in need of treatment. Such vectors are characterized by the further advantage of not requiring host cell proliferation for expression of adenoviral proteins (Horwitz et al., In Virology, Raven Press, New York, vol. 2, pp. 1679-1721 (1990); and Berkner, BioTechniques, 6, 616 (1988)), and, if the targeted tissue for somatic gene therapy is the lung, these vectors have the added advantage of being normally trophic for the respiratory epithelium (Straus, In Adenoviruses, Plenan Press, New York, pp. 451-496 (1984)).
Other advantages of adenoviruses as potential vectors for human gene therapy are as follows: (i) recombination is rare; (ii) there are no known associations of human malignancies with adenoviral infections despite common human infection with adenoviruses; (iii) the adenoviral genome (which is a linear, double-stranded DNA) can be manipulated to accommodate foreign genes that range in size; (iv) an adenoviral vector does not insert its DNA into the chromosome of a cell, so its effect is impermanent and unlikely to interfere with the cell's normal function; (v) the adenovirus can infect non-dividing or terminally differentiated cells, such as cells in the brain and lungs; and (vi) live adenovirus, having as an essential characteristic the ability to replicate, has been safely used as a human vaccine (Horwitz et al. (1990), supra; Berkner et al. (1988), supra; Straus et al. (1984), supra; Chanock et al., JAMA, 195, 151 (1966); Haj-Ahmad et al., J. Virol., 57, 267 (1986); and Ballay et al., EMBO, 4, 3861 (1985)).
The problem of using a recombinant adenovirus in gene therapy is that all cells that express the aforementioned two receptors used by the adenovirus to attach and infect a cell will internalize the gene(s) being administered--not just the cells in need of therapeutic treatment. Likewise, certain cells, such as lymphocytes, which lack the .alpha..sub.v integrin adenoviral receptors, will be severely impaired in the uptake of an adenovirus and will not be easily amenable to adenovirus-mediated gene delivery. Accordingly, limiting adenoviral entry to specific cells and/or expanding the repertoire of cells amenable to adenovirus-mediated gene therapy would be a significant improvement over the current technology. Targeted adenoviral gene delivery should expand the cells amenable to gene therapy, reduce the amount of adenoviral vector that is necessary to obtain gene expression in the targeted cells, as well as reduce side effects and complications associated with increasing doses of an adenovirus, such as inflammation and the transfection of normal, healthy cells.
Attempts have been made to target a virus to specific cells by sterically blocking adenoviral fiber protein with antibodies and chemically linking tissue-specific antibodies to the viral particle (Cotten et al., Proc. Natl. Acad. Sci. USA, 89, 6094-6098 (1992)). Although this approach has demonstrated the potential of targeted gene delivery, the complexity and reproducibility of this approach present major hurdles blocking its application in clinical trials. The difficulties thus far encountered in targeting the virus by these methods involve the method of synthesis required, which is to make major alterations in the viral particles following their purification. These alterations involve additional steps that covalently link large molecules, such as polylysine, receptor ligands and antibodies, to the virus. The targeted particle complexes are not homogeneous in structure and their efficiency is sensitive to the relative ratios of viral particles, linking molecules, and targeting molecules used.
The present invention seeks to overcome the problem of lack of cell and tissue specificity of recombinant adenoviral gene therapy. It is an object of the present invention to provide a receptor-specific, preferably cell receptor-specific/tissue receptor-specific, recombinant adenovirus. A further object of the present invention is to provide means for generating such a recombinant adenovirus at the level of gene expression, thereby enabling purification of recombinant adenoviral particles by conventional techniques. Another object of the present invention is to provide a method of gene therapy involving the use of such a homogeneous adenovirus, without the need for additional components or further modification. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the following detailed description.